Western Blotting for Protein Analysis

Western blotting was conducted as recently published (Neuhaus et al., 2014 (link)). In brief, cells were scraped after the treatments in RIPA buffer on ice. In case of membran protein enrichment, proteins were extracted with 1% Triton-X 100 followed by lysis of the residual proteins in RIPA buffer. All lysis buffers were supplemented with protease inhibitor cocktail and PhosphoSTOP. Protein concentrations were measured by a detergent-compatible Pierce BCA assay (Fisher Scientific). Twenty microgram protein of total lysates or 10 μg of Triton-X 100 and RIPA fractions per lane were loaded onto 7.5, 10, or 12% SDS–PAGE gels. After gel electrophoresis proteins were immunoblotted onto polyvinylidene difluoride membranes. Incubations with primary and secondary antibodies (Supplementary Table S2) were carried out as previously described (Neuhaus et al., 2012b (link)). ECL-solutions were used for the visualization of the bands using a FluorChem FC2 Multiimager II (Alpha Innotech, Hessisch Oldendorf, Germany). Density values of single protein bands were calculated with the software Alpha View and were related to the corresponding β-actin bands.